This application extends work performed during the previous project period, in which we (i) used genome-wide RNAi screens in Drosophila to identify human ORAI1 as a pore subunit of the Ca2+ release-activated Ca2+ (CRAC) channel; (ii) confirmed previous reports that Drosophila Stim and human STIM1 are key regulators of store-operated Ca2+ entry; and (iii) showed that a missense mutation in ORAI1 was responsible for a hereditary severe combined immundeficiency syndrome affecting two human patients. Since then, there has been an explosion of new information on STIM- ORAI interactions. STIM proteins are Ca2+ sensors located in the endoplasmic reticulum (ER), that sense Ca2+ levels in the ER lumen. In response to store depletion, STIM1 and STIM2 oligomerise and move to sites of ER membrane-plasma membrane (ER-PM) apposition where they cause CRAC channels (containing ORAI proteins) to open. Overexpression of STIM and ORAI in a variety of cell types leads to the appearance of large CRAC currents, and expression of a C-terminal fragment of STIM1 can induce the opening of CRAC channels containing ORAI1. Here we will build on these data to extend our understanding of how STIM and ORAI interact to cause CRAC channel opening. In Aim 1, we will define the structural and functional aspects of the STIM1-ORAI1 interaction, and attempt to reconstitute STIM-ORAI signalling in a minimal system in the absence of any other components that might contribute to store-operated Ca2+ entry. In Aim 2, we will use a proteomic approach to identify components of ER membrane-plasma membrane (ER-PM) appositions containing STIM and ORAI, that may functionally modulate STIM-ORAI interactions in mammalian cells. In Aim 3, we will follow up on a genome-wide RNAi screen that we have already performed in mammalian cells, with the objective of identifying new regulators of Ca2+ homeostasis and STIM-ORAI coupling, including mitochondrial regulators such as the mitochondrial uniporter. Together, the proposed experiments should increase our understanding of STIM-ORAI coupling and store-operated Ca2+ entry in several contexts, allowing us to (i) define important structural aspects of the STIM1-ORAI1 interaction; (ii) reconstitute the interaction functionally in minimal systems using recombinant proteins; (iii) identify functional components of larger protein complexes at the plasma membrane of mammalian cells; and (iv) put the interactions into the context of overall Ca2+ homeo- stasis mediated by intracellular proteins and organelles (e.g. mitochondria), again in mammalian cells. The work is broadly relevant to many biological systems, since STIM-ORAI proteins mediate store- operated Ca2+ entry not only in immune/ haematopoietic cells (T and B lymphocytes, mast cells, platelets) but also in skeletal muscle and potentially many other cell types. PUBLIC HEALTH RELEVANCE: No more than 2-3 sentences, describe the relevance of this research to public health. In this section be succinct and use plain language that can be understood by a general, lay audience. Ca2+ is a universal second messenger that is used by all cells, including those of the brain, heart, muscle and immune system, to control a huge diversity of cellular functions. In the previous project period, we identified two essential proteins, STIM and ORAI, that regulate a specialized process of store-operated Ca2+ entry into cells, and showed that mutations in ORAI1 and STIM1 are the underlying cause of at least three instances of hereditary severe combined immunodeficiency in human patients. Here we will use cutting-edge approaches, such as proteomics and genome-wide RNAi screens, to investigate how STIM and ORAI control Ca2+ entry. We expect to identify new regulatory proteins that will provide novel therapies for cancer and autoimmune disease.